Pyrrolysine, a novel amino acid, was recently found to be encoded by UAG codons in a microbial gene for methylamine metabolism. This is the second example of a genetically encoded non-canonical amino acid since selenocysteine, which 18 years ago was found to be encoded by UGA codons in a microbe. Selenocysteine is now known to be widely distributed and the primary form of selenium in humans. Following this precedent, pyrrolysine holds potential of being more widely distributed and of import in metabolism. Regardless, pyrrolysine will serve as a rare example of how organisms can modulate their genetic code to expand metabolic capabilities, an achievement whose replication holds promise for artificially tailoring novel proteins with biomedical potential. Our long-range goals are to understand the function, biogenesis, and genetic encoding of pyrrolysine. Translation of UAG codons is hypothesized to require a specialized UAG decoding tRNA and a dedicated cognate aminoacyl-tRNA synthetase. The roles of these gene products and others thought to be involved in UAG decoding as pyrrolysine will be investigated using biochemical and genetic approaches. The final structure of pyrrolysine is as yet unknown, and insight into pyrrolysine structure and genetic encoding will be gained by analyzing the UAG encoded residue in protein, the cytosol, and on tRNA. An in vivo system has been developed that will be used to study how UAG functions as a sense rather than a stop codon during UAG translation as pyrrolysine. A similar system will be used for site directed mutagenesis in order to test the hypothesized function of pyrrolysine in enzyme catalysis. Finally, transposon mutagenesis will be used to identify unknown genes with anticipated or unanticipated functions during UAG translation as pyrrolysine. [unreadable] [unreadable]